Metabolic flux in biosynthetic pathways endogenous to lactic acid bacteria has been altered for production of products that use pyruvate as a starting substrate. In lactic acid bacteria the major pyruvate metabolic pathway is conversion to lactate through activity of lactate dehydrogenase (LDH). Metabolic engineering to redirect pyruvate from lactate to other products in lactic acid bacteria has had unpredictable results. Production of alanine in LDH-deficient Lactococcus lactis expressing alanine dehydrogenase was shown by Hols et al. (Nature Biotech. 17:588-592 (1999)). However, production of ethanol in LDH-deficient Lactobacillus plantarum expressing pyruvate decarboxylase was very limited, with carbon flow not significantly improved toward ethanol and lactate still produced (Liu et al. (2006) J. Ind. Micro. Biotech. 33:1-7).
In lactic acid bacteria pyruvate is also converted in a pathway to acetolactate, which is then converted to acetoin by acetolactate decarboxylase, and then to 2,3-butanediol. Additional pathways convert acetolactate to diacetyl, valine or leucine. Monnet et al. (Applied and Environmental Microbiology 66:5518-5520 (2000)) have through chemical mutagenesis eliminated acetolactate decarboxylase activity and reduced LDH activity to increase acetolactate, acetoin, and diacetyl production. Disclosed in US Patent Application Publication No. 20100112655 is engineering high flux from pyruvate to 2,3-butanediol in lactic acid bacteria by expressing heterologous butanediol dehydrogenase activity and substantially eliminating lactate dehydrogenase activity.
Disclosed in co-pending US Patent Application Publication No. 2010-0081183 is engineering lactic acid bacteria for high dihydroxy-acid dehydratase (DHAD) activity by expressing a heterologous DHAD and substantially eliminating lactate dehydrogenase activity. DHAD is one of the enzymes in a biosynthetic pathway for synthesis of isobutanol that is disclosed in co-pending US Patent Pub No. US20070092957 A1. Disclosed therein is engineering of recombinant microorganisms for production of isobutanol. Isobutanol is useful as a fuel additive, whose availability may reduce the demand for petrochemical fuels.
Disclosed in de Vos et al. (Int. Dairy J. 8:227-233 (1998)) is that it has appeared impossible to combine inactivation of aldB, encoding acetolactate decarboxylase, with inactivation of ldh, encoding lactate dehydrogenase, in rapidly growing cells of lactic acid bacteria.
There remains a need for altering metabolic flux in lactic acid bacteria away from lactate and away from the acetoin to 2,3-butanediol pathway, and into other biosynthetic pathways downstream of acetolactate, such as for production of isobutanol.